Orthogonal Regulatory Circuits for - ACS Publications - American

Subscriber access provided by Kaohsiung Medical University

Orthogonal regulatory circuits for E. coli based on the #-butyrolactone system of Streptomyces coelicolor Marc Biarnes, Chang-Kwon Lee, Takuya Nihira, Rainer Breitling, and Eriko Takano ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00425 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

1 2

Orthogonal regulatory circuits for E. coli based on the γ-butyrolactone system of Streptomyces coelicolor

3

Author list: Marc Biarnes-Carrera1, Chang-Kwon Lee2,4, Takuya Nihira2,3, Rainer Breitling1

4

and Eriko Takano1

5

Affiliation:

6

1

7

(SYNBIOCHEM), Manchester Institute of Biotechnology, School of Chemistry, Faculty of

8

Science and Engineering, University of Manchester, 131 Princess Street, Manchester M1

9

7DN, United Kingdom.

Manchester

Centre

for

Synthetic

Biology

of

Fine

and

Speciality

Chemicals

10

2

11

565-0871, Japan

12

3

13

Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400,

14

Thailand

15

4

16

Corresponding author: Eriko Takano, [email protected]

17

Graphical Abstract

International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka

Mahidol University-Osaka University Collaborative Research Center for Bioscience and

Present address: Mong-Go Food Co., Ltd., Changwon 641-847, Republic of Korea

18

19

Abstract

20

Chemically inducible transcription factors are widely used to control gene expression of

21

synthetic devices. The bacterial quorum sensing system is a popular tool to achieve such

22

control. However, different quorum sensing systems have been found to cross-talk, both 1 ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 30

23

between themselves and with the hosts of these devices, and they are leaky by nature. Here

24

we evaluate the potential use of the γ-butyrolactone system from Streptomyces coelicolor

25

A3(2) M145 as a complementary regulatory circuit. First, two additional genes responsible

26

for the biosynthesis of γ-butyrolactones were identified in S. coelicolor M145 and then

27

expressed in E. coli BL21 under various experimental conditions. Second, the γ-

28

butyrolactone receptor ScbR was optimised for expression in E. coli BL21. Finally, signal

29

and promoter crosstalk between the γ-butyrolactone system from S. coelicolor and quorum

30

sensing systems from Vibrio fischeri and Pseudomonas aeruginosa was evaluated. The

31

results show that the γ-butyrolactone system does not crosstalk with the quorum sensing

32

systems and can be used to generate orthogonal synthetic circuits.

33

Keywords

34

γ-butyrolactone; Streptomyces; synthetic biology; orthogonal regulatory circuit; quorum

35

sensing

36 37

Synthetic biology aims at the rational design of living organisms with novel functionalities 1.

38

One major challenge is to develop new regulatory circuits that allow for tight regulation over

39

a wide variety of conditions

40

translation rates

41

inducible transcription factors to regulate the expression of genes of interest

42

acyl-homoserine lactones (AHL) from the bacterial quorum sensing (QS) system

6

2,3

. Various tools are available to control transcript levels

or protein concentration

7,8

4,5

,

. One popular tool is the use of chemically3,9

, such as the 3,10

,

11

43

originally derived from the microorganism Vibrio fischerii

44

previously used to generate devices such as oscillators

45

being seen as a promising tool to engineer microbial consortia

46

diversity of well-characterised AHL systems reported. However, despite these promising

47

applications and perspectives, different AHL devices have been found to cross-talk 14, either

48

at the promoter or the signal level

49

unpredictable outcomes. Furthermore, some AHL systems control the expression of virulence

50

factors, such as the one from Pseudomonas aeruginosa 15, and consequently organisms have

51

evolved to target these molecules using so-called quorum quenchers

52

either lactonases or acylases. This can limit the application of QS systems in some hosts, such

53

as mammalian cells

10

12

. This system has been

or logic gates

13

and is currently

10

, mainly due to the large

; a limitation that can result in undesirable or

16

, which function as

17,18

. Finally, the promoter luxp, which drives expression of the output

2 ACS Paragon Plus Environment

Page 3 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


54

signal in the AHL-based synthetic circuits, is leaky 19, resulting in sometimes undesired high

55

basal expression of the output signal.

56

Members of the genus Streptomyces have been widely researched due to their ability to

57

produce a vast array of secondary metabolites, many of which have medical interest

58

Streptomyces, cell-to-cell communication is crucial to coordinate the onset of antibiotic

59

production, as the final compound would be toxic to cells that have not developed the

60

corresponding resistance. This coordination is achieved through the use of small diffusible

61

molecules known as γ-butyrolactones (GBLs)

62

specific

63

Streptomyces species can share a common GBL, currently there is no evidence of signal

64

cross-talk between different GBL regulators at physiological concentrations

65

Streptomyces, GBLs promote a growth phase-dependent switch-like transition to antibiotic

66

production by binding to their cognate receptor protein, a homodimer from the TetR family

67

of repressors. These repressors are the master regulators of biosynthetic gene clusters and in

68

some instances

69

protein.

70

In this study, we evaluated the potential application of the GBL system from S. coelicolor as

71

a regulatory tool for synthetic biology in E. coli. We show that this system can be used as a

72

versatile and accessible tool to activate gene circuits in the heterologous host, with minimal

73

crosstalk with the QS systems from V. fischeri and P. aeruginosa, and that it allows for tight

74

control of genes of interest. To achieve this, we have generated a plasmid for production of

75

GBL SCB2 in E. coli and a series of plasmids constitutively expressing an optimised version

76

of ScbR and a library of Streptomyces-derived promoters: scbRp, scbAp or cpkOp, that are

77

controlling the expression of gfp.

22

. In

21

. These bacterial hormones are species-

, and although some recent publications

26-28

20

23,24

have shown that different 25

. In

they also regulate their own transcription and that of a GBL synthase

78



Page 4 of 30

79

Results and Discussion

80

a) The SCO6264 and SCO6267 genes from S. coelicolor are essential for GBL production

81

In Streptomyces, the biosynthesis of GBLs is reported to start with a condensation between

82

dihydroxyacetone phosphate (DHAP) and a β-ketoacyl-acyl carrier protein (ACP)

83

followed by an intramolecular aldol condensation that yields the corresponding butenolide

84

(Fig. 1, compound 4). In S. coelicolor, the enzyme responsible for this catalytic step is known

85

as ScbA

86

Streptomyces virginiae

87

enzymatically reduced in an NADPH-dependent manner to generate the reduced GBL

88

(compound 5). In some Streptomyces species, this compound undergoes a second enzymatic

89

NADPH-dependent reduction that stereo-specifically reduces the carbonyl in C6 into an (R)-

90

or (S)-alcohol

91

enzymes have been identified and characterised in S. coelicolor. Using a BLASTp search we

92

identified SCO6264 (herafter scbB, 31 % amino acid identity to BarS1

93

ketoacyl-ACP/Coenzyme A (CoA) reductase from the short-chain alcohol dehydrogenase

94

superfamily and SCO6267 (hereafter scbC, 76 % amino acid identity to BprA

26,32

. Previous in vitro studies with homologues from Streptomyces griseus

30

30

29

29

,

and

showed that after this first condensation, the butenolide is

(compound 7). However, to our knowledge, no homologues of these 30

) as a putative 329

and 45 %

33

95

amino acid identity to BarS2 ) as a putative butenolide phosphate reductase.

96

To confirm the involvement of ScbB and ScbC in GBL biosynthesis, insertion mutants of

97

scbB and scbC, LW107 and LW108, respectively, were generated in S. coelicolor M145. The

98

mutant strains were grown in SFM solid medium, their GBLs extracted and analysed using

99

the kanamycin bioassay

34

(Fig. 2). In this assay, only spiking of ethyl acetate extracts

100

resuspended in methanol from strains that produce GBLs will be able to allow growth of

101

reporter strain LW94 in a kanamycin-supplemented medium.

102

Production of GBLs was detected in the wild-type S. coelicolor M145 at all tested extract

103

volumes, using the kanamycin bioassay. However, the indicator strain failed to grow in

104

presence of either LW107 (scbA/C) or LW108 (scbA/B) extracts, suggesting that these

105

mutants are not able to produce the GBLs, confirming that both enzymes are involved in the

106

production of S. coelicolor GBLs.

107 108

b) Expression of the S. coelicolor GBL system in E. coli BL21 results in production of SCB2

109

After identifying the two new genes involved in S. coelicolor GBL production, we

110

constructed a GBL production biosynthesis pathway to confirm their roles and to have an 4 ACS Paragon Plus Environment

Page 5 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


111

easy production platform of GBLs in E. coli. An expression plasmid containing scbA, scbB

112

and scbC under the control of the TetR repressor was constructed. A different TetR-

113

dependent promoter (tetA for scbA 9, Pb10 for scbB and Pb19 for scbC 62) was added in front

114

of each gene, as initial attempts to express the whole operon from a single promoter (tetA)

115

were unsuccessful (data not shown). To facilitate expression of S. coelicolor genes in E. coli

116

BL21, the first ten amino acids of each gene were codon optimised, and were His-tagged.

117

Expression of ScbA and/or ScbC was detected in the soluble and insoluble protein fraction,

118

but not ScbB (Supplementary Fig. S1). In the kanamycin assay (Supplementary Fig. S2),

119

addition of the extract from heterologously expressed ScbA/B/C (pTE1059) in E. coli to the

120

reporter strain induced growth. This extract was then analysed by HPLC-MS/MS (Fig. 3 and

121

Supplementary Fig. S3 and Supplementary Table S4), using an ethyl acetate-methanol extract

122

from S. coelicolor M145 as positive control. In the latter, two peaks are identified in the

123

extracted ion chromatogram (EIC) from the SCB2+Na adduct (theoretical m/z = 267.156678)

124

at a retention time of approximately 19 min after injection. These were assigned as the

125

isomers SCB1 and SCB2, respectively 31,34. In the extract from E. coli, a peak was detected at

126

the same retention time as observed for SCB2 (mass accuracy 0.68 ppm). The identity of the

127

compound identified in the E. coli extract was confirmed through tandem MS showing the

128

same fragmentation pattern as the obtained for SCB1 and SCB2 from S. coelicolor M145

129

(Supplementary Fig. S3). None of the other GBLs produced in S. coelicolor (SCB1 and

130

SCB3) were detected. This can be explained by the fact that the β-ketoacid used in the first

131

catalytic step towards the production of GBLs derives from fatty acid biosynthesis 29,31. Fatty

132

acid biosynthesis starts with the decarboxylative condensation of an acyl-CoA primer with

133

malonyl-acyl carrier protein, catalysed by the gene product of fabH

134

FabH homologue preferentially uses branched acyl-CoA primers for fatty acid production,

135

such as isovaleryl-CoA and isobutyryl-CoA or metylbutyryl-CoA

136

production of both branched (SCB1 and SCB3) and linear (SCB2) GBLs. However, FabH

137

homologue from E. coli has a strong preference of linear acyl-CoA primers, such as acetyl-

138

CoA or malonyl-CoA 38; thus, if the GBL precursors are derived from fatty acid biosynthesis,

139

this would result in exclusive production of linear GBLs (SCB2) in E. coli.

140

To further confirm the role of ScbB and ScbC, single knock-out mutants were generated in

141

the GBL expression plasmid, transformed and expressed in E. coli, and their extracts

142

analysed as described previously (Fig. 3 and Supplementary Fig. S3 and Supplementary

143

Table S4). In the extract from E. coli harbouring scbA/C plasmid (pTE1060)), a peak was

35

. The S. coelicolor

36,37

, which then results in



Page 6 of 30

144

detected in the EIC from the A-factor+Na adduct (theoretical m/z = 265.141028) at

145

approximately 21 min after injection, the same as A-factor from S. griseus (1.93 ppm). In

146

both extracts from S. griseus and E. coli carrying the scbA/C plasmid (pTE1060), an

147

additional broader peak is observed at approximately 20 min after injection. Further analysis

148

through tandem MS showed that both peaks contained fragments consistent with A-factor

149

(Supplementary Fig. S3). In the extract from E. coli carrying scbA/B plasmid (pTE1061),

150

contrary to expectations, no peaks were detected at a mass corresponding to the non-reduced

151

butenolide precursor from A-factor (compound 4), either phosphorylated or not (data not

152

shown). However, an unidentified shunt metabolite was detected at a mass corresponding to

153

A-factor+Na adduct but eluted at an earlier retention time than A-factor. These results show

154

that by heterologous expression of the three GBL biosynthesis genes, GBLs with linear

155

residue can be produced in E. coli.

156 157 158

c) Production of SCB2 in E. coli BL21 is robust through different temperature and medium conditions

159

Synthetic genetic circuits need to be robust under different experimental conditions to allow

160

for predictable rational design of organisms 3. Important parameters to consider are the

161

temperature and the media conditions at which the synthetic organism is grown 39. We tested

162

the qualitative analysis of SCB2 production in E. coli at different temperatures and in four

163

different media. Cells containing the scbA/B/C (pTE1059) plasmid were grown in either M9

164

minimal medium, LB medium, 2xYT medium or terrific broth (TB) medium and in LB at 20,

165

25, 30 and 37 °C. SCB2 production was assayed using the kanamycin bioassay. Production

166

was readily detected at assayed temperatures from 20-30 °C; however, the GBL production

167

was not detected with extracts from cells grown at 37 °C (Fig. 4). This is consistent with

168

previous in vitro enzyme activity results for AfsA

169

activity was found for both enzymes after incubation at and above 35 °C. This could be a

170

potential disadvantage when using the GBL system in E. coli. However, the thermal stability

171

and catalytic activity of the GBL biosynthetic enzymes could be achieved through random

172

mutagenesis, as has been previously shown for other enzymes

173

scbA/B/C was expressed in different media, GBLs were detected in all of the tested

174

conditions, both in minimal and rich media. Therefore, SCB2 production is robust in different

175

experimental conditions.

29

and BarS1

30

, where a reduction in

40,41

. When E. coli harbouring


Page 7 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


176 177

d) Engineering of ScbR to promote solubility in E. coli BL21

178

In S. coelicolor, GBLs promote a switch-like transition to antibiotic production upon binding

179

their cognate receptor. Therefore, after characterising the production of SCB2 in E. coli, we

180

proceeded to characterise the GBL receptor, ScbR, in the heterologous host. Previous reports

181

26,42,43

182

inclusion bodies when expressed in an E. coli background. Furthermore, we, and other groups

183

44

184

homologues

185

obtain a more soluble version of ScbR, we rationally engineered the structure, based on the

186

available structural information. Using the SWISS-MODEL suite

187

ScbR was generated, using CprB crystal structure (PDB 1UI5 48) as template, and visualised

188

using the UCSF Chimera package

189

coelicolor M145 with homology to GBL receptor proteins but unable to bind to the GBLs

190

(pseudo-receptor)

191

identity to CprB) was modelled and compared (Supplementary Fig. S5). As expected, the

192

highly conserved N-terminus of the protein, corresponding to the DNA-binding site of the

193

protein, was similar. However, a hydrophobic patch was identified at the C-terminal end of

194

ScbR, which was not present in CprB. This hydrophobic patch was also not found in the

195

pseudo-receptor ScbR2 (35% amino acid identity to CprB) (Supplementary Fig. S5). To

196

suggest possible roles of this region it was further interrogated using a docking analysis

197

which suggested that it might contain a potential GBL binding site (Supplementary Fig.

198

S6).This suggestion would be consistent with an earlier observation

199

addition of GBLs into a crude extract containing ArpA enhanced the solubility of the protein

200

from inclusion bodies. We therefore hypothesized that GBL receptors might form inclusion

201

bodies due to interaction through this hydrophobic, putatively the GBL-binding, region.

202

Thus, to reduce the aggregation propensity, a 6x arginine tag (Arg6) was added at the C-

203

terminus of the protein. Addition of this tag, either at the N- or the C-terminus, has been

204

shown previously to enhance solubility of proteins without affecting their function 52,53.

205

Recombinant ScbR-Arg6 was expressed from plasmid ScbR-Arg6 + luxp (pTE1067) and its

206

expression was compared to that of recombinant ScbR expressed from ScbR + luxp

207

(pTE1066) by Western Blot analysis (Fig. 5). In this, ScbR-Arg6 in pTE1067 was seen to be

208

more soluble compared to ScbR. To confirm whether the soluble ScbR-Arg6 is functional, a

identified ScbR, and other GBL receptors, as prone to aggregate and form insoluble

, have attempted to crystallise GBL receptors. However, only the structures of distant 45,46

, which are not known to bind GBLs, have been resolved so far. To try to 47

, a model structure of

49

. CprB is a TetR-like DNA-binding protein from S.

22

. The hydrophobic surface of both CprB and ScbR (33% amino acid

51

50

,

that showed that the



Page 8 of 30

209

gel shift assay using protein crude extract was performed (Fig. 5). This shows that addition of

210

increasing amounts of ScbR-Arg6 to DNA, results in appearance of two shifted bands which

211

presumably correspond to complexes of one or two ScbR-Arg6 homodimers bound to the

212

DNA fragment containing operator sequence OR

213

previous reports where ScbR binds its cognate operator sequence as a dimer of homodimers

214

45

215

ScbR-Arg6 resulted in release of the repressor from its cognate operator sequence (see

216

below), whereas it resulted in high variable results when SCB2 was added to untagged ScbR

217

(Supplementary Fig. S7). These results suggest that addition of the Arg6 tag at the C-terminal

218

of ScbR results in less aggregation-prone protein while retaining DNA- and GBL-binding

219

activity.

26

(Fig. 5). This result is consistent with

and previous reported gel shift assays using recombinant ScbR

26

. Addition of SCB2 to

220 221

e) Crosstalk evaluation between the GBL and the AHL systems

222

As previously mentioned, a property that synthetic gene circuits aim to achieve is high

223

orthogonality, that is, that two independent genetic devices do not crosstalk 3. It has been

224

shown that, at physiological conditions, GBLs from Streptomyces only interact with their

225

cognate receptors, although some crosstalk can be achieved at high GBL concentrations,

226

around 200 times higher than the physiological concentration

227

knowledge, it has not been previously explored whether the GBL system is orthogonal to the

228

more well-established signalling system based on the AHLs from V. fisheri and P.

229

aeruginosa. These systems are based on the AHLs binding to LuxR-like proteins. Upon

230

binding to their cognate AHLs, the LuxR receptors act as activators and are able to bind to

231

the corresponding operator sequence (e.g., lux box) and recruit the RNA polymerase to

232

induce transcription of a target gene (Fig. 6). On the other hand, the GBL system is based on

233

the binding of the GBLs to the ScbR-like repressors. The ScbR-like receptor binds the

234

operator sequence (e.g., OR box) and represses the expression of the target gene. Once bound

235

to cognate GBLs, a conformational change occurs to the receptor and can no longer bind to

236

the operator sequence, which results in the activation of the target gene transcript.

237

Signal and promoter crosstalk were evaluated with plasmid vector BC-A1-002

238

derivatives pTE1063 to pTE1069. These plasmids contain either luxR or scbR-Arg6 under the

239

control of a strong constitutive promoter and gfp under the control of the lux promoter (luxp)

240

(BC-A1-002 and pTE1066), the scbA promoter (scbAp) 26 (pTE1064 and pTE1069), the scbR

34,55

. However, to our

54

and


Page 9 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


26

241

promoter (scbRp)

(pTE1063 and pTE1068) or the promoter of the activator of the

242

coelimycin biosynthetic cluster (cpkOp) 56 (pTE1065 and pTE1070). The GFP expression for

243

each construct was measured under different concentrations of 3-oxo-C6-HSL (3OC6), 3-oxo-

244

C12-HSL (3OC12) and purified SCB2, obtained from E. coli supernatant as described in

245

Materials and Methods (Supplementary Fig. S9) to measure the relationship between the

246

input and the response of the synthetic regulatory circuit (Fig. 7, 8).

247

In strains with plasmids containing the luxR, addition of AHLs, either 3OC6 or 3OC12,

248

resulted in activation of the luxp at concentrations above 10-11 M or 10-10 M, respectively, as

249

previously reported

250

systems. However, addition of purified SCB2 only activated luxp at concentrations above 10-

251

6

252

GFP expression was detected upon addition of any of the three signalling molecules when

253

luxR was exchanged for scbR-Arg6. Although we have no clear evidence, this suggests that

254

ScbR-Arg6 does not bind to the operator sequence of LuxR. If ScbR acted as an inducer, one

255

would expect to see a decrease in GFP expression after the addition of SCB2, and if ScbR

256

acted as a repressor, one would expect to see an increase in GFP expression after addition of

257

SCB2, assuming that in both cases the DNA-binding is responsive to the SCB2 signal. We

258

see neither of these occurring.

259

The three ScbR-dependent promoters, scbRp, scbAp and cpkOp, from S. coelicolor, were

260

assayed to evaluate further the potential crosstalk with the AHL system (plasmids pTE1068 –

261

pTE1070). When gfp is placed downstream of any of these promoters in plasmids containing

262

luxR, constitutive expression of GFP was observed, with or without the addition of AHLs or

263

GBL signals. This suggests that the LuxR protein does not interact with any of the tested

264

operator sequences for ScbR, and therefore the promoters are always active (Fig. 7, 8). The

265

GFP expression also corresponded to the strength of the promoters which has been shown

266

before

267

was exchanged for scbR-Arg6, the cells only express gfp at SCB2 concentrations starting from

268

10-9 M, reaching a maximum of induction at 10-7 – 10-6 M. Addition of more SCB2 beyond

269

this concentration resulted in a decrease in gfp expression for scbRp or scbAp (Fig. 7, 8). The

270

potential toxicity of SCB2 in E. coli was discarded as the responsible of this issue, as no

271

decrease in fluorescence was seen in the strains with LuxR and those with cpkOp (Fig. 7, 8)

272

and the final OD600 was independent of the concentration of SCB2 in all samples

273

(Supplementary Fig. S8). This narrow range of activity of the GBL system on these two

10,54

, highlighting the cross-talk potential between the C6 and C12 AHL

M (105 times higher concentration of the signal compared to 3OC6). On the other hand, no

56,57

(e.g., scbRp the strongest promoter, followed by cpkOp and scbAp).When luxR



Page 10 of 30

274

promoters has been previously seen in S. coelicolor, where exogenous addition of SCB1

275

results in precocious antibiotic production only at a narrow range of concentrations 55. Further

276

research on the factors that influence this apparent narrow range of GBL induction would be

277

of great interest. Addition of either AHLs to plasmids containing scbR-Arg6 resulted in no

278

expression of gfp, suggesting that ScbR does not bind to the AHLs and therefore the AHL

279

and GBL signals do not cross-talk at any concentration.

280

These results show that the GBL system from S. coelicolor does not crosstalk with the LuxR

281

system from V. fischerii and that neither the AHLs from V. fischerii nor those from P.

282

aeruginosa bind to ScbR. Furthermore, the GBL system is shown to be highly tightly

283

regulated, resulting in gfp basal expression 4-fold lower than for luxp. This could help create

284

synthetic circuit devices where tight control is required, such as multicellular decision

285

making or multi-state systems

286

containing Streptomyces-derived promoters (scbRp, scbAp and cpkOp) results in relatively

287

low maximum expression of GFP, as opposed to the high levels of expression obtained from

288

luxp, with an approximately 2-fold difference of relative strength between them (Fig. 8). In

289

synthetic microorganisms, especially in those designed to produce speciality chemicals, fine

290

tuning and balancing of all proteins in the biosynthetic pathway has been previously shown to

291

play a crucial role in the titres produced

292

involved in the biosynthetic pathway can result in an impairment of the intracellular

293

metabolite fluxes. The combination of the Streptomyces-derived promoters with the luxp can

294

provide the means to balance and regulate refactored pathways.

21

. Moreover, the response obtained from the plasmids

58,59

, and overly strong induction of the proteins

295 296

f) Induction of the GBL genetic circuits

297

The purification steps to obtain SCB2 in order to activate gene circuits can be costly and

298

time-consuming, and require specialised equipment that may not be accessible to all. To

299

overcome this limitation of the proposed system, we examined whether addition of crude

300

extract containing SCB2, without the previous purification steps, would result in activation of

301

the circuit. GBL producer strain was grown in LB medium under different temperatures, the

302

GBLs were extracted by a simple ethyl acetate extraction of the culture supernatant,

303

resuspended in different volumes of methanol and assayed against a strain containing plasmid

304

pTE1068 (Fig. 9). Expression of GFP was achieved with extracts from cells grown at 20 °C

305

enriched 100-fold, and with extracts from cells grown at 25 and 30 °C enriched 500-fold.


Page 11 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


306

These results show that signalling molecule can be obtained to activate circuits without any

307

need of prior complicated purification steps (e.g., by use of HPLC), making it an accessible

308

and versatile tool.

309

Interestingly, as methanol extracts are added in a 1:100 dilution to the culture with strains

310

containing the reporter plasmid pTE1068, addition of extracts enriched 100-fold corresponds

311

to a concentration of SCB2 similar to that present in the natural supernatant of the producer

312

strain before extraction. This suggests that the whole GBL system (receptor and synthases)

313

could also be used in vivo, albeit some modifications to enhance thermal stability and

314

enzymatic activity might be needed, depending on the application (e.g.; use in human cells).

315

This would then allow to generate complex circuit systems by integrating the whole system

316

inside a single cell to program a determined routine or in different cells (e.g., a sender and a

317

receiver system) to generate a synthetic ecosystems.

318

g) Towards establishing butyrolactone signalling circuits for synthetic biology

319

The generation of novel regulatory circuits that allow the assembly of predictable genetic

320

devices is one of the major challenges of synthetic biology. Currently, AHL-based circuits

321

are one of the popular choices considered when designing such devices. Here, we report the

322

first steps towards the design of the GBL system as a complementary tool for synthetic

323

regulatory circuits. The results presented are the proof-of-concept that the GBL system from

324

Streptomyces can be used in E. coli to control synthetic gene expression. An interesting area

325

of future research will be the further optimisation of the GBL biosynthetic genes through

326

directed evolution to obtain more active and thermally stable versions. This would allow

327

expression of the whole system in mammalian cells, in which GBLs have already been found

328

to be active

329

exploiting the modular nature of the GBL biosynthetic pathway, increasing the amount of

330

orthogonal signal systems available to control gene expression. Finally, the interesting

331

property that addition of high SCB2 concentrations results in inhibition of GFP expression

332

could potentially be used to develop genetic band-pass filters that allow expression of

333

synthetic devices only within a narrow range of inducer concentrations.

60

. Also of great interest would be the diversification of the GBL toolbox by

334



Page 12 of 30

335

Methods

336

a) Bacterial strains and Plasmids.

337

Bacterial strains, oligonucleotides and plasmids used in this study are listed in Supplementary

338

Tables S1-S3. E. coli DH5α strain was used as a host for plasmid construction and

339

maintenance; E. coli BL21 was used for protein expression and GBL production. All

340

oligonucleotides used for PCR amplification and sequencing were synthesized by IDT.

341

To construct a deletion mutant of scbB in S. coelocolor, a 3.6 kb SalI-SphI fragment

342

containing scbB (sco6264) was subcloned into pUC19 to generate pCK1. pCK1 was digested

343

with HincII and then 1.1 kb of thiostrepton-resistance gene (tsr) was inserted. The construct

344

was confirmed to have the desired deletion by DNA sequencing and restriction enzyme

345

digestion. From the resulting plasmid, the BamHI-HindIII fragment was subcloned into the

346

BamHI-HindIII-digested conjugative vector pKC1132 to generate pCK2. The plasmid pCK2

347

was transferred into S. coelicolor by conjugation via E. coli donor ET12567 (pUZ8002) to

348

generate the scbB mutant strain LW107 (∆scbB). A deletion mutant of scbC (sco6267),

349

LW108, in S. coelicolor was constructed by inserting scbC with an apramycin resistance gene

350

and the oriT on cosmid AH10 using the PCR-targeting technology 61. Both the scbB and scbC

351

deletion mutants have been verified by sequencing.

352

To construct plasmid pTE1059 (scbA/B/C), scbA, scbB and scbC were amplified from S.

353

coelicolor M145 genomic DNA using primer pairs scbA_His_5/scbA_3, scbB_5/scbB_His_3

354

and scbC_5/scbC_His_3, respectively. Then these were modified with primer pairs

355

scbA_mod_F/scbA_3, scbB_mod_F/scbB_His_3 and scbC_mod_F/scbC_His_3; and then

356

assembled using Infusion (CloneTech) on plasmid pBbA2k 9, previously digested with EcoRI

357

and XhoI, together with DNA fragments containing promoters Pb10 and Pb19

358

through annealing of primers Pb10_5/3 and Pb19_5/3. Plasmids pTE1060 (scbA/C) and

359

pTE1061 (scbA/B) were constructed by restriction digestion of pTE1059 with NotI and ApaI,

360

respectively, followed by religation.

361

Strain S. coelicolor LW94 was built by integration of reporter plasmid pTE1062, which is

362

plasmid pTE134 34 with scbR in the same orientation as the kanamycin resistant gene (neo).

363

Plasmid BC-A1-002 was a gift from Brian Chow (Addgene plasmid # 78689). To build

364

plasmid pTE1066 or pTE1067, BC-A1-002 was digested with EcoRI and NotI and ligated

365

with EcoRI and NotI digested PCR product containing scbR (primer pair: scbR_5/3) or scbR-

366

Arg6 (primer pair: scbR_5 and scbRarg6_3). Exchange of promoter regions was performed

62

, built


Page 13 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


367

either via whole plasmid amplification, followed by religation (plasmids with scbRp and

368

scbAp) or by ligating a fragment generated through primer annealing to a HindIII and SpeI-

369

digested reporter plasmids (plasmids with cpkOp). All plasmids were confirmed by

370

sequencing.

371

b) Culture media and conditions

372

Luria Broth (LB) medium containing 10 g/L peptone, 5 g/L yeast extract, and 10 g/L NaCl

373

was used for standard bacterial growth and GBL production. GBL input-response relationship

374

was measured using M9 minimal medium (M9) containing 1X M9 salts (Sigma), 0.4 %

375

glucose, 2 mM MgSO4 and 0.1 mM CaCl2, pH 7.0. To evaluate GBL production under

376

different medium, LB and M9 medium were used as previously described; as well as Terrific

377

Broth (TB) containing 12 g/L Tryptone, 24 g/L yeast extract, 0.4 %(v/v) glycerol, and 1X

378

phosphate salts (0.17 M KH2PO4, 0.72 M K2HPO4, pH 7.0); and 2xYT medium (2xYT)

379

containing 16 g/L Bacto Tryptone, 10 g/L Bacto Yeast Extract and 5 g/L NaCl, pH 7.0.

380

Chloramphenicol and kanamycin were supplemented into the media, where appropriate, at

381

concentrations of 30 µg/mL and 50 µg/mL, respectively.

382

c) Expression of the GBL biosynthetic pathway in E. coli

383

Single colonies of E. coli BL21 strains carrying expression plasmids with the GBL synthases

384

(pA15 origin of replication, ~5 copies per cell) were grown at 37 °C for 16 - 18 h in LB

385

supplemented with 50 µg/mL of kanamycin. Samples were diluted to an OD600nm of 0.05 in

386

LB medium without antibiotics and grown at 37 °C until they reached an OD600nm of

387

0.3 - 0.4, when protein expression was induced with 50 nM anhydrotetracycline (aTc) and

388

performed for 5 h at 25 °C. Cells were pelleted at 10,000 xg and 4 °C for 10 min and

389

resuspended in lysis buffer C (150 mM Tris-HCl pH 7.4, 200 mM NaCl and 1 mM DTT,

390

cOmpleteTM tablet (Sigma)) and homogenised by sonication. The soluble protein fraction was

391

recovered after centrifugation at 17,000 xg and 4 °C for 20 min and the insoluble fraction was

392

resuspended in MilliQ grade H2O.

393

When evaluating the robustness of the GBL biosynthetic pathway under different

394

experimental conditions, E. coli cells containing the scbA/B/C (pTE1059) plasmid and

395

originated from the same colony, were grown at 25 °C in either M9 , LB, 2xYT or TB and in

396

LB at 20, 25, 30 and 37 °C.



Page 14 of 30

397

d) GBL extraction and purification

398

Extraction of GBLs from S. coelicolor strains grown in solid cultures was performed as

399

previously described

400

BL21, single colonies of E. coli BL21 strains carrying the corresponding plasmids were

401

grown and expressed as described in the previous section. After centrifugation, the

402

supernatant was mixed with an equal volume of ethyl acetate and vigorously mixed. Organic

403

phase was separated, dried with MgSO4 and the solvent was removed at room temperature

404

and resuspended in 1:100 volume of methanol. In order to purify SCB2, 1.5 L of E. coli

405

BL21/pTE1059 (scbA/B/C) were grown as previously described, with induction at 20 °C for

406

16 - 18 h. Purification was performed on a C18 reverse phase column (Kinetex 5 µm C18 100

407

Ǻ 250 x 21.2 mm) on a preparative HPLC (Agilent 1250 Infinity) equipped with a UV

408

detector set at 210 nm and 254 nm and with a flow rate of 5 mL/min. A maximum volume of

409

5 mL were loaded onto the column and eluted in a linear gradient of 5-100 % methanol + 0.1

410

% formic acid. Samples were collected every minute and subjected to bioassay. Positive

411

samples were pooled, the solvent removed at room temperature and diluted in 5mL of

412

methanol. These were loaded again onto the column and eluted in a linear gradient of 5-100

413

% of acetonitrile + 0.1 % formic acid. Samples were collected every minute and subjected to

414

bioassay. Active samples were pooled, the solvent removed at room temperature and diluted

415

in 5 mL of methanol. This procedure was repeated four times and all samples resuspended in

416

methanol pooled together. The solvent was removed and the samples were weighted,

417

resulting in a total of 15.2 mg of extract obtained. To assess the purity of the sample, the

418

extract was diluted in 1 mL of methanol and subjected to bioassay in serial dilutions of 2-

419

fold. The minimum active dilution was considered to contain 0.05 µg/µL of SCB2, as

420

previously determined by Hsiao et al.

421

resulting in a yield of 2.2 mg/L culture.

422

e) Kanamycin Bioassays

423

Kanamycin bioassays were performed as previously described (63). Briefly, a lawn of S.

424

coelicolor LW94 was prepared in DNAgar plates supplemented with 5 µg/mL of kanamycin

425

by diluting 2.6x106 spores / 100 µL sterile deionised water and evenly spreading across the

426

plate. The plates were allowed to dry for three minutes at room temperature and then 2-3 µL

427

of methanol extract were spotted on the plate and allowed to dry at room temperature. Plates

428

were incubated at 30 °C for 2 - 3 days and growth was monitored every 12 h.

63

. For the extraction of GBL or intermediate metabolites from E. coli

34

. This yielded an active concentration of 3.2 mg,


Page 15 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


429

f) HPLC and MS Analysis

430

A volume of 100 µL of sample (e.g., GBL extract) were diluted 1:2 with HPLC water (total

431

volume 200 µL), centrifuged at 17,000 xg for 15 min to remove any aggregates formed and

432

transferred 150 µL of the resulting product into a glass vial. A total of 15 µL were injected

433

per sample. The solvents used were A: H2O + 0.1% formic acid and B: MeOH + 0.1% formic

434

acid. Both solvents were HPLC grade, from Sigma. The run conditions were 5 min isocratic

435

A:B (95:5) then gradient of 25 min to A:B (5:95), followed by isocratic 5 min A:B (5:95) in

436

an Accucore UHPLC C18 reverse phase column (Thermo Fisher) and a Q Exactive (Thermo

437

Fisher). The ionization conditions were in positive ion mode at a spray voltage of 1.5 kV.

438

g) ScbR expression

439

Expression of ScbR was analysed using E. coli BL21 harbouring plasmids ScbR + luxp

440

(pTE1066) or ScbR-Arg6 + luxp (pTE1067). Single colonies were grown for 16 - 18 h at 37

441

°C in LB medium supplemented with 30 µg/mL of chloramphenicol. Cells were diluted 1:100

442

in LB without antibiotics and grown at 30 °C for 6 h. Cells were centrifuged at 10,000 xg and

443

4 °C for 10 min, the supernatant was discarded and the pellet homogenised by sonication in

444

buffer A

445

soluble protein fraction was recovered from the supernatant after centrifugation for 20 min at

446

17,000 xg and 4 °C and the pelleted insoluble fraction was resuspended in MiliQ grade H2O.

447

h) SDS-PAGE and Western Blot

448

To assess protein expression, crude extracts (either soluble or insoluble fractions) were

449

resolved through SDS-PAGE (10 %(w/v), BioRad) according to Laemmli’s procedure

450

Resolved bands were visualised by Coomassie blue staining (Expedeon). For Western

451

analysis, proteins resolved by SDS-PAGE gels were transferred to a polyvinylidene fluoride

452

(PVDF) membrane by semi-dry blotting. Immunodetection of His-tagged ScbA, ScbB or

453

ScbC was performed using mouse anti-His (Sigma H1029) as primary antibody and IRDye®-

454

conjugated anti-mouse IgG (Abcam ab216772) as secondary antibody, and visualised using

455

LI-COR. Immunodetection of ScbR was carried out using rabbit antiserum raised against

456

ScbR 66 as primary antibody and HRP-conjugated goat anti-rabbit IgG (BioRad) as secondary

457

antibody. The substrate for chemiluminescent detection was Amersham ECL Prime (GE

458

Healthcare) and was visualised using a GeneGnome (Syngene).

64

(50 mM sodium phosphate buffer pH 7.0, cOmpleteTM tablet (Sigma)). The

65

.



Page 16 of 30

459

i) Gel retardation Assays

460

Gel shift assays were performed as previously described 62, using the Roche DIG Gel Shift

461

Kit (Roche). The scbRp was amplified from genomic DNA using primers scbRp_5/3,

462

generating a 144 bp fragment that was labelled with DIG according to manufacturer’s

463

protocol. For each reaction, 25 ng of labelled probe were used. Where appropriate, SCB2

464

extracts were added to the mixture prior incubation. DIG-labelled DNA fragments were

465

immunodetected using antibody mouse anti-DIG (Abcam ab116590) as primary antibody and

466

IRDye®-conjugated anti-mouse IgG (Abcam ab216772) as secondary antibody, and

467

visualised using LI-COR.

468

j) Relationship between signal input and response of GBL and AHL receptors

469

Single colonies of E. coli BL21 cells containing appropriate plasmids were grown for 16 - 18

470

h in LB medium with 30 µg/mL of chloramphenicol and then diluted 1:100 in M9 without

471

antibiotics and grown at 37 °C until an OD600 0.3 - 0.4, where they were aliquoted into 500

472

µL aliquots and supplemented with 1 % (v/v) of the serial dilutions of either 3OC6-HSL

473

(Sigma-Aldrich), 3OC12-HSL (Sigma-Aldrich) or SCB2 (this study) in methanol. Samples

474

were grown at 30 °C for 20 h and then transferred into a 96-well plate in triplicate (150 µL

475

sample/well). OD600 and GFP fluorescence (excitation, 466 nm; emission, 511 nm) were

476

measured in triplicate in a ClarioStar (BMG Labtech) plate reader. Each measurement was

477

performed in biological triplicate.

478 479

k) Structural model of ScbR and docking

480

The model structure of ScbR was generated using the SWISS-MODEL suite

481

crystal structure (PDB 1UI5 48) as template. The resulting structure was exported as PDB file

482

and visualised using the UCSF Chimera package 49. The protein surface was generated using

483

default settings and the hydrophobic regions highlighted with the kdHydrophobicity

484

command line option. Dockings were performed using Autodock Vina software under

485

standard configuration and a grid previously defined with Autodock Tools 50, and were either

486

a broad grid covering the whole protein surface or a more constraint grid covering only the C-

487

terminus end of the protein.

47

and CprB

488


Page 17 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


489

Supporting Information. Table S1: Bacterial strains; Table S2: Plasmids; Table S3:

490

Oligonucleotides; Figure S1: Western Blot analysis of ScbA, ScbB and ScbC protein

491

expression; Figure S2: Kanamycin bioassay to assess production of GBLs in E. coli

492

containing plasmids for the expression of ScbA/B/C; Table S4: MS1 adducts of SCB2 and

493

A-factor; Figure S3: Tandem MS of SCB2 and A-factor; Figure S4: Structural Model of

494

ScbR; Figure S5: Docking results; and Figure S6: Purified SCB2 HPLC-MS analysis.

495 496

Author information

497

Author list:

498

Marc Biarnes-Carrera1, Chang-Kwon Lee2,4, Takuya Nihira2,3, Rainer Breitling1 and Eriko

499

Takano1

500

Affiliation:

501

1

502

(SYNBIOCHEM), Manchester Institute of Biotechnology, School of Chemistry, Faculty of

503

Science and Engineering, University of Manchester, 131 Princess Street, Manchester M1

504

7DN, United Kingdom.

505

2

506

565-0871, Japan

507

3

508

Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400,

509

Thailand

510

4

Manchester

Centre

for

Synthetic

Biology

of

Fine

and

Speciality

Chemicals

International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka

Mahidol University-Osaka University Collaborative Research Center for Bioscience and

Present address: Mong-Go Food Co., Ltd., Changwon 641-847, Republic of Korea

511 512

Author Contributions

513

MBC and CKL performed the experiments. MBC, TN and ET designed the experiments.

514

MBC, TN, RB and ET analysed the results and wrote the manuscript. All authors have read,

515

edited and approved the manuscript.

516

Corresponding Author

517

*E-mail: [email protected]. 17 ACS Paragon Plus Environment


518

ORCID 0000-0002-6791-3256 (Takano)

519

Notes

520

The authors declare no competing financial interest.

Page 18 of 30

521 522

Acknowledgments:

523

MBC was supported by the School of Chemistry, Faculty of Science and Engineering,

524

University of Manchester. Plasmid BC-A1-002 was a gift from Brian Chow. This is a

525

contribution from the Manchester Centre for Synthetic Biology of Fine and Speciality

526

Chemicals (SYNBIOCHEM) and acknowledges the Biotechnology and Biological Sciences

527

Research Council (BBSRC) and Engineering and Physical Sciences Research Council

528

(EPSRC) for financial support (Grant No. BB/M017702/1).

529

530

References

531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561

1. 2. 3. 4. 5. 6. 7.

8. 9.

10. 11. 12. 13.

14.

Khalil, A. S., and Collins, J. J. (2010) Synthetic biology: applications come of age, Nat. Rev. Genetics 11, 367-379. Wang, B., Kitney, R. I., Joly, N., and Buck, M. (2011) Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology, Nat. Commun. 2, 508. Bradley, R. W., Buck, M., and Wang, B. (2016) Tools and Principles for Microbial Gene Circuit Engineering, J. Mol. Biol. 428, 862-888. Davis, J. H., Rubin, A. J., and Sauer, R. T. (2011) Design, construction and characterization of a set of insulated bacterial promoters, Nucleic Acids Res. 39, 1131-1141. Alper, H., Fischer, C., Nevoigt, E., and Stephanopoulos, G. (2005) Tuning genetic control through promoter engineering, Proc. Natl. Acad. Sci. U. S. A. 102, 12678-12683. Salis, H. M., Mirsky, E. A., and Voigt, C. A. (2009) Automated design of synthetic ribosome binding sites to control protein expression, Nat. Biotechnol. 27, 946-950. Andersen, J. B., Sternberg, C., Poulsen, L. K., Bjorn, S. P., Givskov, M., and Molin, S. (1998) New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria, Appl. Environ. Microbiol. 64, 2240-2246. Purcell, O., Grierson, C. S., Bernardo, M., and Savery, N. J. (2012) Temperature dependence of ssrAtag mediated protein degradation, J. Biol. Eng. 6, 10. Lee, T. S., Krupa, R. A., Zhang, F., Hajimorad, M., Holtz, W. J., Prasad, N., Lee, S. K., and Keasling, J. D. (2011) BglBrick vectors and datasheets: A synthetic biology platform for gene expression, J. Biol. Eng. 5, 12. Scott, S. R., and Hasty, J. (2016) Quorum Sensing Communication Modules for Microbial Consortia, ACS Synth. Biol. 5, 969-977. Li, Z., and Nair, S. K. (2012) Quorum sensing: how bacteria can coordinate activity and synchronize their response to external signals?, Protein Sci. 21, 1403-1417. Danino, T., Mondragon-Palomino, O., Tsimring, L., and Hasty, J. (2010) A synchronized quorum of genetic clocks, Nature 463, 326-330. Cornforth, D. M., Popat, R., McNally, L., Gurney, J., Scott-Phillips, T. C., Ivens, A., Diggle, S. P., and Brown, S. P. (2014) Combinatorial quorum sensing allows bacteria to resolve their social and physical environment, Proc. Natl. Acad. Sci. U. S. A. 111, 4280-4284. Wu, F., Menn, D. J., and Wang, X. (2014) Quorum-sensing crosstalk-driven synthetic circuits: from unimodality to trimodality, Chem. Biol. 21, 1629-1638.


Page 19 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619


15. 16. 17. 18.

19. 20.

21. 22. 23.

24.

25. 26.

27.

28.

29.

30.

31.

32.

33.

34.

35. 36.

Smith, R. S., and Iglewski, B. H. (2003) Pseudomonas aeruginosa quorum sensing as a potential antimicrobial target, J. Clin. Invest. 112, 1460-1465. Dong, Y. H., and Zhang, L. H. (2005) Quorum sensing and quorum-quenching enzymes, J. Microbiol. 43, 101-109. Yang, F., Wang, L. H., Wang, J., Dong, Y. H., Hu, J. Y., and Zhang, L. H. (2005) Quorum quenching enzyme activity is widely conserved in the sera of mammalian species, FEBS Lett. 579, 3713-3717. Ozer, E. A., Pezzulo, A., Shih, D. M., Chun, C., Furlong, C., Lusis, A. J., Greenberg, E. P., and Zabner, J. (2005) Human and murine paraoxonase 1 are host modulators of Pseudomonas aeruginosa quorumsensing, FEMS Microbiol. Lett. 253, 29-37. Bansal, K., Yang, K., Nistala, G. J., Gennis, R. B., and Bhalerao, K. D. (2010) A positive feedbackbased gene circuit to increase the production of a membrane protein, J. Biol. Eng. 4, 6. Cimermancic, P., Medema, M. H., Claesen, J., Kurita, K., Wieland Brown, L. C., Mavrommatis, K., Pati, A., Godfrey, P. A., Koehrsen, M., Clardy, J., Birren, B. W., Takano, E., Sali, A., Linington, R. G., and Fischbach, M. A. (2014) Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters, Cell 158, 412-421. Biarnes-Carrera, M., Breitling, R., and Takano, E. (2015) Butyrolactone signalling circuits for synthetic biology, Curr. Opin. Chem. Biol. 28, 91-98. Takano, E. (2006) Gamma-butyrolactones: Streptomyces signalling molecules regulating antibiotic production and differentiation, Curr. Opin. Microbiol. 9, 287-294. Zou, Z., Du, D., Zhang, Y., Zhang, J., Niu, G., and Tan, H. (2014) A gamma-butyrolactone-sensing activator/repressor, JadR3, controls a regulatory mini-network for jadomycin biosynthesis, Mol. Microbiol. 94, 490-505. Recio, E., Colinas, A., Rumbero, A., Aparicio, J. F., and Martin, J. F. (2004) PI factor, a novel type quorum-sensing inducer elicits pimaricin production in Streptomyces natalensis, J. Biol. Chem. 279, 41586-41593. Nodwell, J. R. (2014) Are you talking to me? A possible role for gamma-butyrolactones in interspecies signalling, Mol. Microbiol. 94, 483-485. Takano, E., Chakraburtty, R., Nihira, T., Yamada, Y., and Bibb, M. J. (2001) A complex role for the gamma-butyrolactone SCB1 in regulating antibiotic production in Streptomyces coelicolor A3(2), Mol. Microbiol. 41, 1015-1028. Lee, K. M., Lee, C. K., Choi, S. U., Park, H. R., Kitani, S., Nihira, T., and Hwang, Y. I. (2005) Cloning and in vivo functional analysis by disruption of a gene encoding the gamma-butyrolactone autoregulator receptor from Streptomyces natalensis, Arch. Microbiol. 184, 249-257. Kinoshita, H., Ipposhi, H., Okamoto, S., Nakano, H., Nihira, T., and Yamada, Y. (1997) Butyrolactone autoregulator receptor protein (BarA) as a transcriptional regulator in Streptomyces virginiae, J. Bacteriol. 179, 6986-6993. Kato, J. Y., Funa, N., Watanabe, H., Ohnishi, Y., and Horinouchi, S. (2007) Biosynthesis of gammabutyrolactone autoregulators that switch on secondary metabolism and morphological development in Streptomyces, Proc. Natl. Acad. Sci. U. S. A. 104, 2378-2383. Shikura, N., Yamamura, J., and Nihira, T. (2002) barS1, a gene for biosynthesis of a gammabutyrolactone autoregulator, a microbial signaling molecule eliciting antibiotic production in Streptomyces species, J. Bacteriol. 184, 5151-5157. Sidda, J. D., Poon, V., Song, L., Wang, W., Yang, K., and Corre, C. (2016) Overproduction and identification of butyrolactones SCB1-8 in the antibiotic production superhost Streptomyces M1152, Org. Biomol. Chem. 14, 6390-6393. Hsiao, N. H., Soding, J., Linke, D., Lange, C., Hertweck, C., Wohlleben, W., and Takano, E. (2007) ScbA from Streptomyces coelicolor A3(2) has homology to fatty acid synthases and is able to synthesize gamma-butyrolactones, Microbiology 153, 1394-1404. Lee, Y. J., Kitani, S., Kinoshita, H., and Nihira, T. (2008) Identification by gene deletion analysis of barS2, a gene involved in the biosynthesis of gamma-butyrolactone autoregulator in Streptomyces virginiae, Arch. Microbiol. 189, 367-374. Hsiao, N. H., Nakayama, S., Merlo, M. E., de Vries, M., Bunet, R., Kitani, S., Nihira, T., and Takano, E. (2009) Analysis of two additional signaling molecules in Streptomyces coelicolor and the development of a butyrolactone-specific reporter system, Chem. Biol. 16, 951-960. Lai, C. Y., and Cronan, J. E. (2003) Beta-ketoacyl-acyl carrier protein synthase III (FabH) is essential for bacterial fatty acid synthesis, J. Biol. Chem. 278, 51494-51503. Choi, K. H., Heath, R. J., and Rock, C. O. (2000) beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis, J. Bacteriol. 182, 365-370.



620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677

37.

38.

39. 40. 41.

42.

43. 44. 45.

46.

47. 48.

49.

50. 51.

52. 53. 54.

55.

56.

57.

Page 20 of 30

Li, Y., Florova, G., and Reynolds, K. A. (2005) Alteration of the fatty acid profile of Streptomyces coelicolor by replacement of the initiation enzyme 3-ketoacyl acyl carrier protein synthase III (FabH), J. Bacteriol. 187, 3795-3799. Tsay, J. T., Oh, W., Larson, T. J., Jackowski, S., and Rock, C. O. (1992) Isolation and characterization of the beta-ketoacyl-acyl carrier protein synthase III gene (fabH) from Escherichia coli K-12, The J. Biol. Chem. 267, 6807-6814. Cardinale, S., and Arkin, A. P. (2012) Contextualizing context for synthetic biology--identifying causes of failure of synthetic biological systems, Biotechnol. J. 7, 856-866. Song, J. K., and Rhee, J. S. (2000) Simultaneous enhancement of thermostability and catalytic activity of phospholipase A(1) by evolutionary molecular engineering, Appl. Environ. Microbiol. 66, 890-894. Soh, L. M. J., Mak, W. S., Lin, P. P., Mi, L., Chen, F. Y., Damoiseaux, R., Siegel, J. B., and Liao, J. C. (2017) Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design, ACS Synth. Biol. 6, 610-618. Yang, Y. H., Kim, T. W., Park, S. H., Lee, K., Park, H. Y., Song, E., Joo, H. S., Kim, Y. G., Hahn, J. S., and Kim, B. G. (2009) Cell-free Escherichia coli-based system to screen for quorum-sensing molecules interacting with quorum receptor proteins of Streptomyces coelicolor, Appl. Environ. Microbiol. 75, 6367-6372. Kudo, N., Kimura, M., Beppu, T., and Horinouchi, S. (1995) Cloning and characterization of a gene involved in aerial mycelium formation in Streptomyces griseus, J. Bacteriol. 177, 6401-6410. Horinouchi, S., and Beppu, T. (2007) Hormonal control by A-factor of morphological development and secondary metabolism in Streptomyces, Proc. Jpn. Acad., Ser. B Phys. Biol. Sci. 83, 277-295. Bhukya, H., Bhujbalrao, R., Bitra, A., and Anand, R. (2014) Structural and functional basis of transcriptional regulation by TetR family protein CprB from S. coelicolor A3(2), Nucleic Acids Res. 42, 10122-10133. Mohd-Sharif, N., Shaibullah, S., Givajothi, V., Tan, C. S., Ho, K. L., Teh, A. H., Baharum, S. N., Waterman, J., and Ng, C. L. (2017) Crystallization and X-ray crystallographic analysis of recombinant TylP, a putative gamma-butyrolactone receptor protein from Streptomyces fradiae, Acta Crystallogr., Sect. F: Struct. Biol. Commun. 73, 109-115. Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006) The SWISS-MODEL workspace: a webbased environment for protein structure homology modelling, Bioinformatics 22, 195-201. Natsume, R., Ohnishi, Y., Senda, T., and Horinouchi, S. (2004) Crystal structure of a gammabutyrolactone autoregulator receptor protein in Streptomyces coelicolor A3(2), J. Mol. Biol. 336, 409419. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., and Ferrin, T. E. (2004) UCSF Chimera--a visualization system for exploratory research and analysis, J. Comput. Chem. 25, 1605-1612. Trott, O., and Olson, A. J. (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading, J. Comput. Chem. 31, 455-461. Onaka, H., Nakagawa, T., and Horinouchi, S. (1998) Involvement of two A-factor receptor homologues in Streptomyces coelicolor A3(2) in the regulation of secondary metabolism and morphogenesis, Mol. Microbiol. 28, 743-753. Fuchs, S. M., and Raines, R. T. (2005) Polyarginine as a multifunctional fusion tag, Protein Sci. 14, 1538-1544. Kato, A., Maki, K., Ebina, T., Kuwajima, K., Soda, K., and Kuroda, Y. (2007) Mutational analysis of protein solubility enhancement using short peptide tags, Biopolymers 85, 12-18. Magaraci, M. S., Bermudez, J. G., Yogish, D., Pak, D. H., Mollov, V., Tycko, J., Issadore, D., Mannickarottu, S. G., and Chow, B. Y. (2016) Toolbox for Exploring Modular Gene Regulation in Synthetic Biology Training, ACS Synth. Biol. 5, 781-785. Takano, E., Nihira, T., Hara, Y., Jones, J. J., Gershater, C. J., Yamada, Y., and Bibb, M. (2000) Purification and structural determination of SCB1, a gamma-butyrolactone that elicits antibiotic production in Streptomyces coelicolor A3(2), J. Biol. Chem. 275, 11010-11016. Takano, E., Kinoshita, H., Mersinias, V., Bucca, G., Hotchkiss, G., Nihira, T., Smith, C. P., Bibb, M., Wohlleben, W., and Chater, K. (2005) A bacterial hormone (the SCB1) directly controls the expression of a pathway-specific regulatory gene in the cryptic type I polyketide biosynthetic gene cluster of Streptomyces coelicolor, Mol. Microbiol. 56, 465-479. Gottelt, M., Kol, S., Gomez-Escribano, J. P., Bibb, M., and Takano, E. (2010) Deletion of a regulatory gene within the cpk gene cluster reveals novel antibacterial activity in Streptomyces coelicolor A3(2), Microbiology 156, 2343-2353


Page 21 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703


58.

59.

60.

61.

62. 63.

64.

65. 66.

Ajikumar, P. K., Xiao, W. H., Tyo, K. E., Wang, Y., Simeon, F., Leonard, E., Mucha, O., Phon, T. H., Pfeifer, B., and Stephanopoulos, G. (2010) Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli, Science 330, 70-74. Sung, M., Yoo, S., Jun, R., Lee, J., Lee, S., and Na, D. (2016) Optimization of phage lambda promoter strength for synthetic small regulatory RNA-based metabolic engineering, Biotechnol. Bioproc. E 21, 483-490. Weber, W., Schoenmakers, R., Spielmann, M., El-Baba, M. D., Folcher, M., Keller, B., Weber, C. C., Link, N., van de Wetering, P., Heinzen, C., Jolivet, B., Sequin, U., Aubel, D., Thompson, C. J., and Fussenegger, M. (2003) Streptomyces-derived quorum-sensing systems engineered for adjustable transgene expression in mammalian cells and mice, Nucleic Acids Res. 31, e71. Gust, B., Challis, G.L., Fowler, K., Kieser, T., and Chater, K.F. (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin, Proc. Natl. Acad. Sci. U. S. A. 100,1541-1546. Cox, R. S., Surette, M. G., and Elowitz, M. B. (2007) Programming gene expression with combinatorial promoters, Mol. Syst. Biol. 3, 145. Biarnes-Carrera, M., Breitling, R., and Takano, E. (2018) Chapter 10. Detection and quantification of butyrolactones from Streptomyces. In Quorum Sensing: Methods and Protocols, Methods in Molecular Biology (Leoni, L., and Rampioni, G., Eds.), In Press, vol. 1673, DOI: 10.1007/978-1-4939-73095_10. Natsume, R., Takeshita, R., Sugiyama, M., Ohnishi, Y., Senda, T., and Horinouchi, S. (2003) Crystallization of CprB, an autoregulator-receptor protein from Streptomyces coelicolor A3(2), Acta Crystallogr., Sect. D: Biol. Crystallogr. 59, 2313-2315. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227, 680-685. Gottelt, M., Hesketh, A., Bunet, R., Puri, P., and Takano, E. (2012) Characterisation of a natural variant of the gamma-butyrolactone signalling receptor, BMC Res. Notes 5, 379.

704



705

Page 22 of 30

Figure 1

706 707 708 709 710 711 712 713 714 715 716 717 718 719 720

Figure 1. Schematic representation of the GBL biosynthesis gene cluster from S. coelicolor A3(2) M145 and the possible biosynthetic pathway leading towards production of GBLs: SCB1, SCB2 or SCB3 in S. coelicolor; based on the biosynthetic pathways proposed by Kato et al. (29) for A-factor production in S. griseus and by Shikura et al. (30) for virginiae butanolides in S. virginiae. The biosynthesis of GBLs has been proposed to start with the condensation of DHAP with a β-ketoacid by (29, 31) ScbA in S. coelicolor A3(2), leading to product 3, which putatively undergoes a spontaneous intramolecular Claisen condensation, resulting in butenolide 4. This compound is reduced by a butenolide phosphate reductase, ScbC, yielding 5, which could be hydrolysed, yielding compound 8. The phosphate group is lost, resulting in the A-factor-like GBL 6, which can also be hydrolised to render the open lactone 9. The final GBLs (7) are obtained after stereo-specific reduction through a 3-ketoacyl-ACP/CoA reductase, ScbB. During transition phase, GBLs accumulate in the environment and bind to the receptor ScbR, resulting in a switch-like transition towards antibiotic production.


Page 23 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

721


Figure 2

722

723 724 725 726 727 728

Figure 2. Kanamycin bioassay plates with different concentrations (1 µL, 2.5 µL, 5 µL and 10 µL) of GBL ethyl acetate extracts from the different Streptomyces strains as depicted above the plates. Growth in the presence of kanamycin is detected in all concentrations of S. coelicolor M145 extract, whereas no growth is seen for extracts from LW107 (scbA/C) and LW108 (scbA/B) mutants, suggesting these genes are involved in the production of GBLs.

729



730

Page 24 of 30

Figure 3

731 732 733 734 735 736 737 738 739 740 741 742 743 744 745

Figure 3. Analysis of intermediate metabolites in GBL biosynthesis through LC-MS. Extracted Ion Chromatograms (EIC) of extracts from E. coli cells expressing pTE1059 (scbA/B/C), pTE1060 (scbA/C) or pTE1061 (scbA/B). (A) When expressing the three genes using pTE1059, a EIC peak is detected that elutes at the same retention time as SCB2 from S. coelicolor M145 extract. This peak was further shown to be SCB2 by MS/MS analysis (Supplementary Fig. S3). (B) After deleting scbB in pTE1060, this peak is no longer detectable; instead two peaks are detected at a mass corresponding to A-factor, which elute at the same retention time as the two peaks of A-factor from S. griseus, suggesting that scbB is responsible for the stereospecific reduction of compound 6. (C) Deletion of scbC in pTE1061 results in an unidentified detectable peak at a mass corresponding to A-factor, which elutes at a distinctive retention time. The concentration of the anhydrotetracycline (aTc) and the positive control extracts from S. coelicolor or S. griseus is denoted at the side. (D) Overlap of peaks identified in E. coli ethyl acetate extracts showed in panels A - C, highlighting the distinctive retention time between them.

746 24 ACS Paragon Plus Environment

Page 25 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

747


Figure 4

748 749 750 751 752 753 754 755

Figure 4. Qualitative production of SCB2 at different temperatures and media. Production of SCB2 in E. coli was assessed under different temperatures from 20 °C to 37 °C and different media conditions, minimal to rich media. (A) Kanamycin bioassay with extracts from E. coli producer cells grown at different temperatures. Expression of SCB2 is detected at assayed temperatures from 20 – 30 °C, but not at 37 °C. (B) Kanamycin bioassay with extracts from E. coli harbouring scbA/B/C (pTE1059) grown under different media. All media allowed production of SCB2.

756 757



758

Page 26 of 30

Figure 5

759

760 761 762 763 764 765 766 767 768 769 770

Figure 5. Western Blot and gel retardation analysis of ScbR-Arg6.. (A) Western Blot analysis of ScbR and ScbR-Arg6 in ScbR + luxp plasmid (pTE1066) and ScbR-Arg6 + luxp plasmid (pTE1067), respectively, at stationary phase in the soluble and insoluble fractions, and compared to plasmid BC-A1-002, which does not contain ScbR. A faint band corresponding to ScbR (black arrow) can be seen in pTE1066 soluble fraction, which is stronger in pTE1067. Addition of the Arginine tag improved the solubility of ScbR. (B) Gel retardation analysis of ScbR-Arg6 against a DNA fragment containing the operator sequence OR (26). Addition of increasing amounts of ScbR-Arg6 results in formation of complex ScbRArg6:OR and 2 ScbR-Arg6:OR, showing that addition of the Arg6 tag does not affect DNAbinding properties of ScbR.

771


Page 27 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

772


Figure 6

773 774 775 776 777 778 779 780 781 782 783 784

Figure 6. Signal and promoter crosstalk between the AHL and the GBL systems: constructs. Schematic representation of the plasmids built to evaluate signal and promoter crosstalk between the AHL and the GBL systems. In plasmid BC-A1-002 (54), constitutive expression of the luxR results in production of LuxR, which binds to the LUX box when the concentration of AHLs reaches a concentration threshold. There, it induces gfp expression by RNA polymerase recruitment. Exchange of the luxp and the LUX box from BC-A1-002 for scbRp and ScbR cognate operator site (OR) generated plasmid pTE1063, used to evaluate whether LuxR could interact with ScbR OR. Parallel construction of pTE1067 was created by replacing luxR for scbR-Arg6 and was used to evaluate whether ScbR could interact with the LUX box. Finally, plasmid pTE1058 replaced both the luxp by scbRp and luxR by scbR-Arg6 and should respond only to the addition of SCB2.

785



786

Page 28 of 30

Figure 7

787 788 789 790 791 792 793 794 795 796 797 798 799 800 801

Figure 7. Signal and promoter crosstalk between the AHL and the GBL systems. Normalised GFP/OD600 output after addition of different concentrations of autoinducer concentrations ([AI]/M), either 3OC6HSL (blue line), 3OC12HSL (green line) or SCB2 (red line); to strains harbouring plasmids BC-A1-002 (LuxR+luxp), pTE1063 (LuxR+scbRp), pTE1067 (ScbR-Arg6+luxp) and pTE1068 (ScbR-Arg6+scbRp) (Fig 6). Measurements were made ~20 h after induction and growth at 30 °C. Expression of gfp is at the maximum with concentrations of 10−9 M of 3OC6-HSL and 10−8 M of 3OC12-HSL, when added to BC-A1002. However, no GFP expression is seen when AHLs are added to the cells with the ScbRArg6+luxp (pTE1067). On the other hand, addition of any signalling molecules results in no change in gfp expression in LuxR+scbRp (pTE1063), where scbRp is always active. GFP expression is induced upon the addition of SCB2 with the ScbR-Arg6+scbRp (pTE1068). Interestingly, addition of an excess of SCB2 in ScbR-Arg6+scbRp results in repression of the system. No induction of GFP was observed with addition of AHLs to ScbR-Arg6+scbRp (pTE1068).

802 28 ACS Paragon Plus Environment

Page 29 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

803


Figure 8

804 805 806 807 808 809 810 811 812 813 814 815 816

Figure 8. Expression of GFP with different ScbR-dependent promoters results in a gradient of output signal. Normalised GFP/OD600 output after addition of different concentrations of autoinducer ([AI]/M) 3OC6HSL (blue line) or SCB2 (red line); to strains harbouring pTE1063 (LuxR + scbRp), pTE1064 (LuxR + scbAp), pTE1065 (LuxR + cpkOp), pTE1068 (ScbR-Arg6 + scbRp), pTE1069 (ScbR-Arg6 + scbAp) or pTE1070 (ScbR-Arg6 + cpkOp). Measurements were made ~20 h after induction and growth at 30 °C. As seen in Fig 7, plasmids containing LuxR are active at all concentrations of signals, suggesting that LuxR does not interact with any of the ScbR operator sequences to repress the promoters. Exchange of luxR for scbR-Arg6 results in repression of the promoters until around 10−9 M of SCB2. Interestingly, both scbRp and scbAp are only active in concentrations between 10-8 to 10-4 Mof SCB2, respectively. However, cpkOp is active at all concentrations above 10−9 M, and the activity was not shut down.

817



818

Page 30 of 30

Figure 9

819 820 821 822 823 824 825 826

Figure 9. Response analysis of plasmid pTE1068 under different extracts obtained from 10 mL LB culture of E. coli producer cells. The results match the previously observed characteristics, where lower temperatures seem to be favourable for SCB2 production (Fig. 3). Interestingly, addition of a 100-fold concentrated ethyl acetate extracts of the liquid culture supernatant to the reporter cells (shaded box) resulted in full activation of gfp expression at 20 °C and about half activation of gfp expression at 25 and 30 °C, suggesting that the system could be used at different temperatures to fine tune gene expression.

827 828


Orthogonal Regulatory Circuits for - ACS Publications - American

Recommend Documents